1. Field of the Invention
The present invention generally concerns (1) an apparatus, as are commonly but not exclusively used in laboratories, for illuminating and for viewing a macroscopically-sized specimen, for example a mouse, along a viewing axis, and (2) the methods of using such an apparatus.
The present invention particularly concerns viewing, along a single viewing axis at a single time, a macroscopically-sized specimen panoramically, and over more than one hundred and eighty degrees of viewing angle about the specimen. The specimen may be viewed panoramically in and by its reflection of the illuminating light(s) and/or, more commonly, also in such fluorescent emissions as are induced in the specimen by the illuminating light(s). “Macroscopically-sized” means only that the specimen is not microscopic, and may be observed by the naked eye.
The present invention also particularly concerns image illumination for presenting along a single viewing axis at a single time multiple views, each view along an associated axis, of a specimen. Axial illumination along each axis may be separately independently adjusted so that the resulting image view is selectively balanced in either of intensity and/or color (i.e., wavelength, or frequency). The image views may in particular show fluorescence induced in the specimen (along each, and all, illumination axis) at more than one color (i.e., wavelength, or frequency). These fluorescent image regions of differing colors can be controlled so as to appear in a composite image with roughly equal intensity.
Moreover, and further particularly, the selective illumination along each of multiple axis in accordance with the present invention will be seen to support calibration of the observed intensity of fluorescence at each different color (i.e., wavelength, or frequency), as may occur along each and all viewing axis
Still further, and finally, the present invention will be seen to concern a “racetrack”, or “cattle chute”, direction of live specimens, for example, mice into a viewing area where each may be automatically simultaneously (1) illuminated along multiple axis, and panoramically observed along multiple viewing axis, while (2) illumination along each axis such as induces multi-colored fluorescence in the specimen will be balanced so that image regions of different fluorescent coloration are themselves balanced, while (3) the true intensity, and color (wavelength, or frequency) of all images, and image views, and image portions may be quantitatively known.
2. Description of the Prior Art
2.1 General Laboratory Apparatus and Methods for Illumination and Observation of Macroscopically Sized Specimens
Apparatus to illuminate and to hold macroscopically-sized specimens for viewing, including viewing as may involve the taking of photographs, are known in the art. These apparatus hold secure a macroscopically-sized specimen to be viewed, including for example a live specimen and more particularly a laboratory animal and still more particularly a mouse, upon a specimen stage. One or more sources of illuminating radiation, most commonly light radiation, are brought to bear upon the held specimen.
The illuminating radiation sources may consist of the emitting end of a fiber optic, a fiber optic bundle, or a light pipe or the like. The illuminating radiation itself may, by way of example, be sufficient so as to induce fluorescence in the specimen, including in a specimen as may have been previously fused with fluorescing agents that most commonly serve to make regions of the specimen that are of interest more visible or otherwise detectable.
The illuminated specimen may be, and commonly is, digitally imaged, but may also and/or alternatively be photographed, including in its emitted fluorescent light.
There images so formed are basically nothing but crude snapshots, normally adjusted only in overall intensity and this only by intervention of the image taker, or researcher, such as by adjustment of the intensity of illumination(s), or the exposure of the camera. These images leave a great deal to be desired. Quite commonly a specimen may be infused with multiple fluorescing agents, or dyes, and/or so that different regions of the specimen fluoresce at different colors, or so that the same region of the specimen, such as growing tumor, will exhibit fluorescence of different colors in differing (but often overlapping) areas resultantly to having been infused with a different fluorescing agent (i.e., dyed) at different times (during the growth of the tumor). Some fluorescent colors in some image regions may be bright and/or extensive, obscuring less bright and/or less extensive regions of other colors nonetheless that all image regions—both bright and dim—are of equal observational significance.
Furthermore, there is commonly no way to calibrate the brightness of the induced fluorescence(s).
Finally, the images are not produced automatically even though an image camera may have any of auto-focus and/or auto-exposure. This is because the composite, multi-color fluorescent image presented to the camera is really an image that must have its intensity (brightness), and/or its color, adjusted in parts, and not (normally) as a whole—which is all that an automatic camera is capable of doing. According that the composite images have not been automatically produced, applicant knows of no attempt in the prior art to automatically position a succession of specimens for automatic imaging, such as, in particular, photographing a succession of live mice.
As background to the present invention, the general nature of imaging, and of photographing, macroscopically-sized specimens is contained in paper #3658-12, Panoramic epifluorescent visualization of cardiac action potential activity, pp. 99-107 by Mark A. Bray, Vanderbilt Univ., Nashville, Tenn., USA; Marc Lin, Vanderbilt Univ., Nashville, Tenn., USA; John P. Wikswo, Jr., Vanderbilt Univ., Nashville, Tenn., USA This paper is available, circa 2004, at the Vanderbilt University web sites of one or more of its authors.
2.2 The Utility of Introducing Quantitative Rigor into Observations of Macroscopically Sized Specimens
The present and related inventions will generally be seen to be directed to a common goal of imparting the imaging, and photographing, of macroscopic specimens (especially specimens as are caused to fluoresce)—a process generally presently conducted “ad hoc”—with a great deal of scientific rigor.
As of present, circa 2004, the images, or photographs produced by conventional illumination and observation of macroscopically-sized specimens, such as the biological specimen of a mouse, tend to be rather crude. Most typically the mouse will be illuminated so that an region of interest, such as a tumor, previously absorbing fluorescent dye will be caused to fluoresce, and the fluorescent region of the resulting image is indicated only that the mouse has the tumor.
In this rudimentary observation many, many things are lacking.
First, it is not possible to view the mouse specimen along multiple axis, or panoramically around a broad angular field, at the same time. This precludes looking at the same tumor in the mouse from two or more different directions, and from looking at multiple tumors as may exist within different regions of the mouse all at the same time.
Accordingly, it would firstly be useful if a single macroscopically-sized specimen, for example a mouse, could be observed along each of multiple axis, for example left side and right side and fore and aft, all at the same time.
Second, no dimensional scale, either linear or grid, typically accompanies the viewed image of the specimen (the mouse). Such a scale is useful for, by way of example, judging the dimension(s) and volume of the observed tumor. Accordingly, it would secondly be useful if the image of a specimen (for example, a mouse) inherently contained a scale of either the linear or the grid type.
Third, and although it is common for a single specimen to contain multiple fluorescing agents which fluoresce at different colors so as to identify corresponding regions of interest within the (single) specimen, it is not commonly thought to attempt adjustment of the intensity of each color within a resulting composite image. In other words, a body impregnated with fluorescent green dye may appear to fluoresce green light quite brightly while another body (or the same body or portion thereof as may have picked up red fluorescent dye at a different time and/or to a different extent) may, under the same common illumination, fluoresce red light quite dimly. Nonetheless that the body, or tumor, fluorescing green shows brightly in the image, and the body, or tumor, fluorescing red shows but dimly in the image, the “green” tumor or stage may be of equal size and/or interest to the “green” tumor. What looks bright, and what looks dim, in the composite image is, of course, a function of the efficiency of the uptake of the fluorescent dyes, the efficiency of the illumination excitation of each, and the efficiency of each dye to fluoresce, among other factors.
Although not common, it is, of course, possible to use multiple illumination sources of different intensities and/or frequencies, independently adjusting selecting illumination frequencies and adjusting the intensity of each so that the resulting “green” tumor and “green” tumor images in the composite are somewhat comparable. The related invention for CONTROLLED-INTENSITY MULTIPLE-WAVELENGTH MULTIPLE-AXIS ILLUMINATION OF MACROSCOPIC SPECIMENS FROM A SINGLE LIGHT SOURCE FROM SPECIAL MASKS, FILTERS AND/OR BIFURCATED CABLES will be seen to deal in an elegant way with this challenge of exciting fluorescent emissions of different colors so that the differently colored areas of a composite image appear comparable. The viewing apparatus of the present invention will be seen to be fully compatible with the related invention, which uses bifurcated cables. However, the viewing apparatus of the present invention will also be seen to be fully suitable for use with multiple light sources that are independently controlled in color and/or, most commonly, intensity.
Accordingly, it would thirdly be useful if each of multiple regions fluorescing at different colors within a single composite image of a specimen (for example, a mouse) could be independently adjusted in intensity, clearly rendering visible in the composite image those things and/or regions that the researcher and image taker desires to be well seen, while suppressing within the composite image other things and/or regions that are deemed unimportant. It would be especially useful if this selective differential “highlighting” of each of multiple colors of fluorescent emission could somehow be realized from but a single, common, illuminating light source.
Some little thought will reveal, however, that should such control be given to the image maker, then it may become impossible to know what has been done in manipulation of the composite image and its several colors, and to know what imaged things and/or regions “really” look like under “normal”, or known predetermined, conditions. It is thus problematic that no scale of the intensity(ies) and/or colors (i.e., wavelengths, or frequencies) of (potentially several different) fluorescent emission(s) typically accompanies the viewed image of the specimen (the mouse). Such an intensity and/or color would be scale is useful for, by way of example, judging how bright or how dim, or of exactly what color(s), were things and/or regions appearing in the composite image—nonetheless that the appearance of things or regions within this composite image may have been enhanced, or attenuated in intensity, or may even be “off-shade” in color (i.e., wavelength, or frequency)—under normal, standard, and predetermined illumination conditions.
Accordingly, it would fourthly be useful if the image of a specimen (for example, a mouse) inherently contained a scale of by which any of the intensity(ies), color(s), or, as even more exotic criteria seldom useful, radiation temperature might be accurately known. The color scale might be broken down into hue, chroma (purity, or saturation) and brightness (value). In this manner a viewer of a composite image might be able to say: “I see by comparison to a scale that is within the selfsame image that this clearly visible first object (or area) fluoresced green, and that it was in fact quite bright, even to the point of obscuration, until intentionally diminished in intensity. Meanwhile I also see by comparison to another portion of the same scale, or another scale also contained within the image, that this equally clearly visible second object (or area) fluoresced green, but only dimly so, and that this second object has intentionally been accentuated in intensity by action of the image maker. In fact, by comparison to the scale, I can quantitatively determine the absolute quantitative brightness (i.e., intensity) of each and all of the green and the red fluorescing regions. Still further, I can see by comparison to this same scale that both the colors green are red are in hue (i.e., wavelength, or frequency) precisely as would be expected as emissions from their respective fluorescent dyes. I must thus assume that there is no extraneous colored light falling upon the specimen, and that I am looking at a true and accurate image of fluorescent emissions from the specimen, without anything else or any extraneous color contamination of this image.”
Fifth, it would be useful if, in addition to making better and more comprehensive images bearing improved quantitative information, if it were possible to produce such images with a greater degree of automation. It would in particular be useful if in the imaging of large numbers of live animals, such as mice, the successive images of successive mice—no matter how widely differing in their fluorescing tumors or the like—could not only be automatically correctly quantitatively recorded, image after image, but if the mice could be “herded” past an imaging point, with each mouse in turn being automatically imaged.